1. Field of the Invention
This invention relates to a novel vaccine specific for Staphylococcus aureus, the major bacterium responsible for bovine mastitis. The strains of the invention are used in vaccine development, to protect individuals from S. aureus infection and to treat and control S. aureus-induced mastitis.
2. Description of the Relevant Art
S. aureus mastitis affects 90% of US. Dairy herds. Annual loss due to mastitis in the United States is two billion dollars (Nickerson et al. 1984. J. Dairy Res. 51: 209–217). Antibiotic treatment of S. aureus infections has met with limited success due to the development of antibiotic resistant strains and their ability to survive within polymorphonuclear neutrophils (PMN) and macrophages where very few antibiotics achieve effective intracellular concentrations (Craven and Anderson. 1984. J. Dairy Res. 51:513–523). S. aureus mastitis is rarely acute, but causes subclinical, chronic infections (Bramley and Dodd. 1984. J. Dairy Res. 51:481–512). Cows are most susceptible to S. aureus infections during the transition from lactation to involution and from involution to colostrogenesis (Oliver and Sordillo. 1988. J. Dairy Sci. 71: 2584–2606). The bovine udder has numerous defense mechanisms to protect against invading pathogens.
The first line of defense against bacterial invasion of the udder is the smooth muscle sphincter surrounding the teat end (Frost, A. J. 1990. In: Proc. Int. Symp. Bovine Mastitis, Page 1) and keratin, a waxy material found in the teat canal (Murphy, J. M. 1959. Cornell Vet. 49: 411–421). However, bacteria can breach the teat canal and enter the gland cistern by multiplication or reverse flow during milking machine pulsation (Nickerson, S. C. 1986. In: Dairy Research Report, Louisiana Agric. Experiment Station, Page 211; Schalm et al. 1971. In: Bovine Mastitis, Lea and Febiger, Philadelphia, Pa., Page 209).
Once inside the gland, the second line of defense against invading pathogens is phagocytosis by neutrophils and macrophages. Macrophages are the predominant cell type in uninfected lactating glands, but neutrophils are recognized as the most important phagocytic cell because of their rapid migration from blood during an intramammary infection and because of their efficient phagocytosis of bacteria (Paape et al. 1979. J. Dairy Sci. 62: 135–153). Neutrophils migrate from blood to milk in response to chemoattractants produced by bacteria and the host during the inflammatory process. Once inside the gland cistern, neutrophils must rely on random collisions to bring them into contact with invading organisms. Large numbers of neutrophils (9×105 neutrophils/ml) are required to provide effective protection. However, this concentration far exceeds the number of neutrophils actually found in the healthy gland (<1×105 neutrophils/ml).
However, phagocytosis by neutrophils and macrophages present in the milk can be enhanced by specific antibodies (immunoglobulins (Ig), opsonins) which act to reduce the number of neutrophils needed to resist infection. Antibodies bind to bacteria via the Fab region of the Ig. Neutrophils attach to bacteria via surface receptors for the Fc portion of Ig and ingest the bacteria. Such bacterial attachment via Fc receptors promotes both ingestion and digestion of the organism. Bovine neutrophils have Fc receptors for IgG2 and bovine mammary macrophages have Fc receptors for IgG1. However, once inside the gland, S. aureus form an exopolysaccharide capsule that is itself low in immunogenicity and also serves to block Ig Fab from recognizing the highly antigenic cell-wall proteins it encapsulates. Thus, stimulation of the production of specific opsonins to S. aureus is inhibited by this formation of an exopolysaccharide capsule, a fact having a distinct bearing on the composition required for a vaccine against S. aureus. 
Once ingested, bacteria are contained in a vacuole (phagosome). Phagosomes fuse with lysosomal granules to form phagolysosomes which together contain oxygen-dependent and oxygen-independent antibacterial mechanisms and enzymes that kill and digest the organisms. Unfortunately, phagocytosis and killing of S. aureus is not always complete (Craven et al. 1984. J. Dairy Res. 51: 513–523) and the S. aureus that survive are capable of multiplication, increasing the chances for establishing chronic infections. However, phagocytized S. aureus are flushed out during milking.
Should the S. aureus escape phagocytosis, they have the potential to adhere to the epithelial lining of the gland, the first step in the colonization in the udder (Frost, A. J. 1975. Infect. Immun. 12:1154–1156; Wanasinghe, D. D. 1981. Acta Vet Scand. 22: 109–117). Growth in milk increases the ability of S. aureus to adhere to epithelial cells (Mamo et al. 1994. Microbiol. Immunol. 38: 305–308). Organisms adhere to the epithelium lining the gland cistern and milk ducts and form small colonies that become covered with an exopolysaccharide capsule (Nickerson, S. C. 1993. In: Proc. Reg. Meeting Natl. Mastitis Council, Syracuse, N.Y., Page 64). Toxins produced by adhered S. aureus damage the underlying epithelium. Eventually, S. aureus penetrate the epithelium and establish deep-seated abscesses that become walled off by scar tissue. These abscesses are impenetrable by antibiotics and therefore result in chronic subclinical infections (Guterbock, W. M. 1992. Vet Med. X: 1229–1234).
Different mastitis pathogens have different target cell specificities and use different mechanisms to adhere to cells of the bovine mammary gland (Lammers et al. 2001. Vet. Microbio. 80(3): 255–265). The mechanism(s) for S. aureus adherence to epithelial cells is not clearly understood, but the host receptors collagen and fibronectin have been identified (Cifrian et al. 1994. J. Dairy Sci. 77: 970–983; Cifrian et al. 1996. Vet. Microbiol. 48: 187–198; Cifrian et al. Am. J. Vet. Res. 57: 1308–1311; Dziewanowska et al. 1999. Infect. Immun. 67: 4673–4678; Foster, T. J. 1991. Vaccine 9: 221–227; Foster and Hook. 1998. Trends in Microbiol. 6: 484–488; Hensen et al. 2000. J. Dairy Sci. 83: 418–429; Joh et al. 1994. Biochem. 33: 6086–6092; Joh et al. 1999. Matrix Biol. 18: 211–223; Mohamed et al. 1999. Infect. Immun. 67: 589–594). Fibronectin-binding proteins are present on the cell wall of the majority of S. aureus isolates and appear to be a major player in bacterial adherence (Lammers et al. 1999. FEMS Microbiol. LETT. 180: 103–109). However, the exact mechanism of fibronectin adherence has not been defined (Joh et al., 1999, supra). In addition to facilitating adherence, this interaction has been shown to induce a host protein that inhibits phagocytosis (Jonsson and Wadstrom. 1993. In: Pathogenesis of Bacterial Infections in Animals, Gyles and Thoen, Eds., Iowa State University Press, Ames, Iowa, Page 21). Recent studies demonstrated that specific antibody to staphylococcal fibronectin-binding protein blocked tissue adherence and enhanced phagocytosis (Mamo et al. 1995. Microb. Pathog. 19: 49–55; Cifrian et al. 1994. J. Dairy Sci. 77: 970–983). S. aureus have also been shown to bind to collagen and laminin (Cifrain et al. 1996, Am. J. Vet Res., supra) which can also be blocked by specific antibodies (Lorca et al. 2002. FEMS Microbiol. Lett 206: 31–37).
Attempts have been made to produce antibodies which are specific for S. aureus and which will enhance phagocytosis. Such attempts have focused on the changing surface properties of the organisms once inside the gland (Aguilar et al. 2001. Vet. Microbiol. 82(2): 165–175; Lorca et al., supra; Nickerson et al., 1993, supra. Norcross et al. 1983. Nordhaug et al. J. Dairy Sci. 77: 1267–1275; Nordhaug et al. J. Dairy Sci. 77: 1276–1284; Olmsted et al. 1992. Infect. Immun. 60: 249–256). Though S. aureus form an exopolysaccharide capsule upon entering the mammary gland, there is a period of time during the logarithmic growth phase that S. aureus are unencapsulated, thus exposing the highly antigenic cell-wall proteins. Adherence proteins are among the cell-wall proteins, which makes this a critical phase in the S. aureus invasion of the gland. This is the phase during which both opsonizing and anti-adherent antibodies are most effective. Upon reaching the stationary phase of the growth cycle in vivo, S. aureus produce an exopolysaccharide capsule. The exopolysaccharide capsule is low in imunogenicity; and furthermore, there are multiple capsule serotypes, thus increasing the difficulty of generating opsonizing antibodies.
Numerous attempts have been made to produce vaccines that induce protective antibodies against S. aureus. These attempts have met with varying degrees of success. These have included whole cell, cell lysates, toxoids, live attenuated organisms, and inactivated organisms (Brock et al. 1975. Res. Vet Sci. 19: 152–158; Buzzola et al. 2001. Epidemiol. Infect. 126(3): 445–452; Calzolare et al. 1997. J. Dairy Sci. 80: 854–858; Derosa et al. 1997. Zentralbl. Veterinarmed. [B] 44: 599–607; Giraudo et al. 1997. J. Dairy Sci. 80: 845–853; Herbelin et al. 1997. J. Dairy Sci. 80: 2025–2034; O'Brien et al. 2000. J. Dairy Sci. 83:1758–1766; O'Brien et al. 2001. J. Dairy Sci. 84: 1791–1799; Tyler et al. 1993. Vet. Clin. North Am. Food Anim. Pract. 9: 537–549; Watson, D. L. 1984. J. Dairy Sci. 67: 2608–2613; Watson, D. L. 1992. Res. Vet. Sci. 53: 346–353; Watson et al. 1996. Aust. Vet. J. 74: 447–450; Yoshida et al. 1984. J. Dairy Sci. 67: 620–627; Zecconi et al. 1999. J. Dairy Sci. 82: 2101–2107). Varying serotypes have been a major factor in the limited success of these vaccines (Hensen et al., supra; Sordelli et al. 2000. J. Clin. Microbiol. 38: 846–850; Tollersrud et al. 2000. J. Clin. Microbiol. 38: 2998–3003). The importance of using the appropriate serotype in producing a S. aureus vaccine is evident by the improved success of vaccines prepared from individual herd isolates (autogenous vaccines; Hwang et al. 2000. J. Vet Med. Sci. 62: 875–880; Sears et al. 1999. Natl. Mastitis Council Annual Meeting, National Mastitis Council, Inc., Pages 86–92). Where autogenous vaccines have failed could be attributed to variation of serotypes within a herd (Guidry et al. 1997. Vet Microbiol. 59: 53–58; Hoedemaker et al. 2001. J. Vet Med. B Infect. Dis. Vet Public Health 48: 373–383).
Thus, there is a need for agents useful for the immunotherapy of S. aureus-induced mastitis which would prevent or limit the disease.